Receptor for the F4 fimbriae of enterotoxigenic Escherichia coli (ETEC).

Infection with F4(+) enterotoxigenic Escherichia coli (ETEC) responsible for diarrhea in neonatal and post-weaned piglets leads to great economic losses in the swine industry. These pathogenic bacteria express either of three fimbrial variants F4ab, F4ac, and F4ad, which have long been known for their importance in host infection and initiating protective immune responses. The initial step in infection for the bacterium is to adhere to host enterocytes through fimbriae-mediated recognition of receptors on the host cell surface. A number of receptors for ETEC F4 have now been described and characterized, but their functions are still poorly understood. The current review summarizes the latest research addressing the characteristics of F4 fimbriae receptors and the interactions of F4 fimbriae and their receptors on host cells. These include observations that as follows: (1) FaeG mediates the binding activities of F4 and is an essential component of the F4 fimbriae, (2) the F4 fimbrial receptor gene is located in a region of chromosome 13, (3) the biochemical properties of F4 fimbrial receptors that form the binding site of the bacterium are now recognized, and (4) specific receptors confer susceptibility/resistance to ETEC F4 infection in pigs. Characterizing the host-pathogen interaction will be crucial to understand the pathogenicity of the bacteria, provide insights into receptor activation of the innate immune system, and develop therapeutic strategies to prevent this illness.

Antibody repertoire development in fetal and neonatal piglets. XX. B cell lymphogenesis is absent in the ileal Peyer's patches, their repertoire development is antigen dependent, and they are not required for B cell maintenance.

The continuous ileal Peyer's patches (IPP) of sheep are regarded as a type of mammalian bursal equivalent where B cells diversify their repertoire in an Ag-independent fashion. Anatomically and developmentally similar IPP occur in swine. Resection of ∼90% of the IPP in piglets at birth did not alter Ig levels in serum and secretions or retard diversification of the Ab repertoire when animals were maintained in isolators and colonized with a defined gut flora. Resection or sham surgery elevated IgG and IgA in serum and in lavage fluid from the gut, lung, and in saliva. No changes in the frequency of IgG-, IgA-, and IgM-containing cells in the spleen and peripheral lymph node were observed. Using an index that quantifies diversification of the VDJ repertoire, no differences were seen in three secondary lymphoid tissues between piglets lacking IPP and colonized controls, whereas both groups displayed >10-fold greater diversification than did late-term fetal piglets or piglets maintained germ-free. Somatic hypermutation was very low in fetal IPP and the IPP of germ-free piglets but increased 3- to 5-fold after colonization. D-J signal joint circles were not recovered in IPP, and V-DJ signal joint circles were 5-fold lower than in bone marrow and similar to those in thymus and spleen. We conclude that the porcine IPP are not a site of B cell lymphogenesis, do not undergo Ag-independent repertoire diversification, and are not primary lymphoid tissue since they are not required for maintenance of Ig levels in serum and secretions.

The lack of an inherent membrane targeting signal is responsible for the failure of the matrix (M1) protein of influenza A virus to bud into virus-like particles.

The matrix protein (M1) of influenza A virus is generally viewed as a key orchestrator in the release of influenza virions from the plasma membrane during infection. In contrast to this model, recent studies have indicated that influenza virus requires expression of the envelope proteins for budding of intracellular M1 into virus particles. Here we explored the mechanisms that control M1 budding. Similarly to previous studies, we found that M1 by itself fails to form virus-like-particles (VLPs). We further demonstrated that M1, in the absence of other viral proteins, was preferentially targeted to the nucleus/perinuclear region rather than to the plasma membrane, where influenza virions bud. Remarkably, we showed that a 10-residue membrane targeting peptide from either the Fyn or Lck oncoprotein appended to M1 at the N terminus redirected M1 to the plasma membrane and allowed M1 particle budding without additional viral envelope proteins. To further identify a functional link between plasma membrane targeting and VLP formation, we took advantage of the fact that M1 can interact with M2, unless the cytoplasmic tail is absent. Notably, native M2 but not mutant M2 effectively targeted M1 to the plasma membrane and produced extracellular M1 VLPs. Our results suggest that influenza virus M1 may not possess an inherent membrane targeting signal. Thus, the lack of efficient plasma membrane targeting is responsible for the failure of M1 in budding. This study highlights the fact that interactions of M1 with viral envelope proteins are essential to direct M1 to the plasma membrane for influenza virus particle release.

Genetic fusions of heat-labile (LT) and heat-stable (ST) toxoids of porcine enterotoxigenic Escherichia coli elicit neutralizing anti-LT and anti-STa antibodies.

Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal disease in humans and farm animals. E. coli fimbriae, or colonization factor antigens (CFAs), and enterotoxins, including heat-labile enterotoxins (LT) and heat-stable enterotoxins (ST), are the key virulence factors in ETEC diarrhea. Unlike fimbriae or LT, STa has not often been included as an antigen in development of vaccines against ETEC diarrhea because of its poor immunogenicity. STa becomes immunogenic only after being coupled with a strongly immunogenic carrier protein. However, native or shorter STa antigens either had to retain toxic activity in order to become antigenic or elicited anti-STa antibodies that were not sufficiently protective. In this study, we genetically mutated the porcine LT (pLT) gene for a pLT(192(R-->G)) toxoid and the porcine STa (pSTa) gene for three full-length pSTa toxoids [STa(11(N-->K)), STa(12(P-->F)), and STa(13(A-->Q))] and used the full-length pLT(192) as an adjuvant to carry the pSTa toxoid for pLT(192):pSTa-toxoid fusion antigens. Rabbits immunized with pLT(192):pSTa(12) or pLT(192):pSTa(13) fusion protein developed high titers of anti-LT and anti-STa antibodies. Furthermore, rabbit antiserum and antifecal antibodies were able to neutralize purified cholera toxin (CT) and STa toxin. In addition, preliminary data suggested that suckling piglets born by a sow immunized with the pLT(192):pSTa(13) fusion antigen were protected when challenged with an STa-positive ETEC strain. This study demonstrated that pSTa toxoids are antigenic when fused with a pLT toxoid and that the elicited anti-LT and anti-STa antibodies were protective. This fusion strategy could provide instructive information to develop effective toxoid vaccines against ETEC-associated diarrhea in animals and humans.

Correction: Moxley, R.A., et al. Intimate Attachment of Escherichia coli O157:H7 to Urinary Bladder Epithelium in the Gnotobiotic Piglet Model. Microorganisms 2020, 8, 263.

The authors wish to make the following corrections to this paper [...].

Intimate Attachment of Escherichia coli O157:H7 to Urinary Bladder Epithelium in the Gnotobiotic Piglet Model.

Enterohemorrhagic Escherichia coli (EHEC), a pathogenic subset of Shiga toxin-producing E. coli (STEC), is an important cause of hemorrhagic colitis and hemolytic-uremic syndrome (HUS), and a rare cause of urinary tract infections (UTIs) with associated HUS. EHEC strains attach intimately to intestinal epithelium with formation of actin pedestals (attaching-effacing (A/E) lesions); however, the mechanism of EHEC attachment to the uroepithelium is unknown. We conducted a retrospective study on archived urinary bladder specimens from gnotobiotic piglets that naturally developed cystitis associated with EHEC O157:H7 infection following oral inoculation and fecal shedding. Paraffin-embedded bladder tissues from three piglets with cystitis and immunohistochemical evidence of EHEC O157:H7 adherence to the uroepithelium were processed for and examined by transmission electron microscopy. EHEC O157:H7 bacteria were found in one of three piglets, intimately attached to pedestals on the apical surfaces of the superficial urothelium (umbrella cells). Cystitis was significantly associated with the length of survival of the piglets post-inoculation (p = 0.0339; estimated odds ratio = 2.6652). This is the first report of E. coli causing A/E-like lesions in the uroepithelium, and also evidence of the utility of the gnotobiotic piglet as a model for studies of the pathogenesis of EHEC UTIs.

Heat-labile enterotoxin promotes Escherichia coli adherence to intestinal epithelial cells.

Given recent evidence suggesting that the heat-labile enterotoxin (LT) provides a colonization advantage for enterotoxigenic Escherichia coli (ETEC) in vivo, we hypothesized that LT preconditions the host intestinal epithelium for ETEC adherence. To test this hypothesis, we used an in vitro model of ETEC adherence to examine the role of LT in promoting bacterium-host interactions. We present data demonstrating that elaboration of LT promotes a significant increase in E. coli adherence. This phenotype is primarily dependent on the inherent ADP-ribosylation activity of this toxin, with a secondary role observed for the receptor-binding LT-B subunit. Rp-3',5'-cyclic AMP (cAMP), an inhibitor of protein kinase A, was sufficient to abrogate LT's ability to promote subsequent bacterial adherence. Increased adherence was not due to changes in the surface expression of the host receptor for the K88ac adhesin. Evidence is also presented for a role for bacterial sensing of host-derived cAMP in promoting adherence to host cells.

Characterization of the binding specificity of K88ac and K88ad fimbriae of enterotoxigenic Escherichia coli by constructing K88ac/K88ad chimeric FaeG major subunits.

Enterotoxigenic Escherichia coli (ETEC) strains expressing K88 (F4) fimbriae are the major cause of diarrhea in young pigs. Three antigenic variants of K88 fimbriae (K88ab, K88ac, and K88ad) have been identified among porcine ETEC strains. Each K88 fimbrial variant shows a unique pattern in binding to different receptors on porcine enterocytes. Such variant specificity in fimbrial binding is believed to be controlled by the major subunit (FaeG) of the K88 fimbriae, because the genes coding for the only other fimbrial subunit are identical among the three variants. Uniqueness in binding to host receptors may be responsible for differences in the virulence levels of porcine diarrhea disease caused by K88 ETEC strains. To better understand the relationships between the structure of FaeG proteins and fimbrial binding function, and perhaps virulence in disease, we constructed and expressed various K88ac/K88ad faeG gene chimeras and characterized the binding activity of each K88 chimeric fimbria. After verifying biosynthesis of the chimeric fimbriae, we examined their binding specificities in bacterial adherence assays by using porcine brush border vesicles that are specific to either the K88ac or K88ad fimbria. Results showed that each fimbria switched binding specificity to that of the reciprocal type when a peptide comprising amino acids 125 to 163 was exchanged with that of its counterpart. Substitutions of a single amino acid within this region negatively affected the binding capacity of each fimbria. These data indicate that the peptide including amino acids 125 to 163 of the FaeG subunit is essential for K88 variant-specific binding.

Toxin-mediated effects on the innate mucosal defenses: implications for enteric vaccines.

Recent studies have confirmed older observations that the enterotoxins enhance enteric bacterial colonization and pathogenicity. How and why this happens remains unknown at this time. It appears that toxins such as the heat-labile enterotoxin (LT) from Escherichia coli can help overcome the innate mucosal barrier as a key step in enteric pathogen survival. We review key observations relevant to the roles of LT and cholera toxin in protective immunity and the effects of these toxins on innate mucosal defenses. We suggest either that toxin-mediated fluid secretion mechanically disrupts the mucus layer or that toxins interfere with innate mucosal defenses by other means. Such a breach gives pathogens access to the enterocyte, leading to binding and pathogenicity by enterotoxigenic E. coli (ETEC) and other organisms. Given the common exposure to LT(+) ETEC by humans visiting or residing in regions of endemicity, barrier disruption should frequently render the gut vulnerable to ETEC and other enteric infections. Conversely, toxin immunity would be expected to block this process by protecting the innate mucosal barrier. Years ago, Peltola et al. (Lancet 338:1285-1289, 1991) observed unexpectedly broad protective effects against LT(+) ETEC and mixed infections when using a toxin-based enteric vaccine. If toxins truly exert barrier-disruptive effects as a key step in pathogenesis, then a return to classic toxin-based vaccine strategies for enteric disease is warranted and can be expected to have unexpectedly broad protective effects.

Development and characterization of swine primary respiratory epithelial cells and their susceptibility to infection by four influenza virus types.

Influenza viruses are a group of respiratory pathogens that have evolved into four different types: A, B, C, and D. A common feature is that all four types are capable of replicating and transmitting among pigs. Here, we describe the development of isogenous cell culture system from the swine respiratory tract to study influenza viruses. Phenotypic characterization of swine primary nasal turbinate, trachea and lung cells revealed high expression of cytokeratin and demonstrated tissue site dependent expression of tight junction proteins. Furthermore, lectin binding assay on these cells demonstrated higher levels of Sia2-6Gal than Sia2-3Gal receptors and supported the replication of influenza A, B, C, and D viruses to appreciable levels at both 33 and 37 °C, but replication competence was dependent on virus type or temperature used. Overall, these swine primary respiratory cells showed epithelial phenotype, which is suitable for studying the comparative biology and pathobiology of influenza viruses.

Pre-exposure with influenza A virus A/WSN/1933(H1N1) resulted in viral shedding reduction from pigs challenged with either swine H1N1 or H3N2 virus.

There is an urgent need to develop a broad-spectrum vaccine that can effectively prevent or eliminate the spread of co-circulating swine influenza virus strains in multiple lineages or subtypes. We describe here that pre-exposure with a live virus generated via a A/WSN/1933(H1N1) reverse genetics system resulted in a significant reduction of viral shedding from pigs exposed to either a swine H1N1 virus or a swine H3N2 virus. At 3-day post challenge (DPC), approximately 1 log and 1.5 logs reductions of viral shedding were observed in the swine H1N1- and H3N2-challenged vaccinated pigs when compared to unvaccinated animals. A further decline in viral load was observed at 5 DPC where viral shedding was decreased by greater than 3 logs in vaccinated pigs receiving either the H1N1 or H3N2 virus challenge. Although the sera of the vaccinated pigs contained high titers of neutralizing antibodies against the vaccine strain, measured by Hemagglutination Inhibition (HI) assay, only suboptimal HI titers of neutralizing antibody were detected in the post-challenge serum of the vaccinated animals using the challenge swine H1N1 virus. The substantial genetic and antigenic differences between the vaccine virus and the challenge viruses imply that the observed protection may be mediated by mechanisms other than neutralization by IgG, such as non-neutralizing antibody activities, mucosal immunity, or conserved T cell immunity, which warrants further investigation.

Comparison of the contributions of heat-labile enterotoxin and heat-stable enterotoxin b to the virulence of enterotoxigenic Escherichia coli in F4ac receptor-positive young pigs.

In swine, the most common and severe enterotoxigenic Escherichia coli (ETEC) infections are caused by strains that express K88 (F4)(+) fimbriae, heat-labile enterotoxin (LT), heat-stable enterotoxin b (STb), and enteroaggregative E. coli heat-stable toxin 1. Previous studies based on a design that involved enterotoxin genes cloned into a nontoxigenic fimbriated strain have suggested that LT but not STb plays an important role in dehydrating diarrheal disease in piglets <1 week old and also enhances bacterial colonization of the intestine. In the present study, we compared these two toxins in terms of importance for piglets >1 week old with a study design that involved construction of isogenic single- and double-deletion mutants and inoculation of 9-day-old F4ac receptor-positive gnotobiotic piglets. Based on the postinoculation percent weight change per h and serum bicarbonate concentrations, the virulence of the STb(-) mutant (Delta estB) did not significantly differ from that of the parent. However, deletion of the LT genes (Delta eltAB) in the STb(-) mutant resulted in a complete abrogation of weight loss, dehydration, and metabolic acidosis in inoculated pigs, and LT complementation restored the virulence of this strain. These results support the hypothesis that LT is a more significant contributor than STb to the virulence of F4(+) ETEC infections in young F4ac receptor-positive pigs less than 2 weeks old. However, in contrast to previous studies with gnotobiotic piglets, there was no evidence that the expression of LT enhanced the ability of the F4(+) ETEC strain to colonize the small intestine.

Escherichia coli constructs expressing human or porcine enterotoxins induce identical diarrheal diseases in a piglet infection model.

To develop a piglet model for studying diarrheal disease and developing vaccines, we challenged gnotobiotic piglets with isogenic Escherichia coli strains constructed to express porcine 987P(F6) fimbriae and a heat-labile or a heat-stable enterotoxin to examine clinical outcomes. Piglets developed identical diarrheal diseases when inoculated with constructs expressing human or porcine enterotoxins.

Detailed mapping of the linear B Cell epitopes of the hemagglutinin (HA) protein of swine influenza virus.

Using the Hemagglutinin (HA) protein peptide array of H1N1 pdm09 and a panel of swine antisera against various swine influenza H1 and H3 clusters, we identified three immunoreactive epitopes with one (peptide 15) located in HA1 (amino acids 57-71) and two (peptides 121 and 139) in HA2 (amino acids 481-495 and 553-566). Further analysis showed that all swine antisera of H1 clusters efficiently recognized two HA2 epitopes; peptides 121 and 139, with only a subset of antisera reactive to HA1-derived peptide 15. Interestingly, none of these peptides were reactive to SIV H3 antisera. Finally, intranasal inoculation of peptides 15 and 121 into pigs revealed that peptide 121, not peptide 15, was able to generate antibody responses in some animals. The results of our experiments provide an important foundation for further analyzing the immune response against these peptides during natural viral infection and also provide peptide substrates for diagnostic assays.

Comparison of Porcine Airway and Intestinal Epithelial Cell Lines for the Susceptibility and Expression of Pattern Recognition Receptors upon Influenza Virus Infection.

Influenza viruses infect the epithelial cells of the swine respiratory tract. Cell lines derived from the respiratory tract of pigs could serve as an excellent in vitro model for studying the pathogenesis of influenza viruses. In this study, we examined the replication of influenza viruses in the MK1-OSU cell line, which was clonally derived from pig airway epithelium. MK1-OSU cells expressed both cytokeratin and vimentin proteins and displayed several sugar moieties on the cell membrane. These cells also expressed both Sial2-3Gal and Sial2-6Gal receptors and were susceptible to swine influenza A, but not to human B and C viruses. Interestingly, these cells were also permissive to infection by influenza D virus that utilized 9-O-acetylated glycans. To study the differences in the expression of pattern recognition receptors (PRRs) upon influenza virus infection in the respiratory and digestive tract, we compared the protein expression of various PRRs in MK1-OSU cells with that in the SD-PJEC cell line, a clonally derived cell line from the porcine jejunal epithelium. Toll-like receptor 7 (TLR-7) and melanoma differentiation-associated protein 5 (MDA5) receptors showed decreased expression in influenza A infected MK1-OSU cells, while only TLR-7 expression decreased in SD-PJEC cells. Further research is warranted to study the mechanism behind the virus-mediated suppression of these proteins. Overall, this study shows that the porcine respiratory epithelial cell line, MK1-OSU, could serve as an in-vitro model for studying the pathogenesis and innate immune responses to porcine influenza viruses.

Efficacy of Urtoxazumab (TMA-15 Humanized Monoclonal Antibody Specific for Shiga Toxin 2) Against Post-Diarrheal Neurological Sequelae Caused by Escherichia coli O157:H7 Infection in the Neonatal Gnotobiotic Piglet Model.

Enterohemorrhagic Escherichia coli (EHEC) is the most common cause of hemorrhagic colitis and hemolytic uremic syndrome in human patients, with brain damage and dysfunction the main cause of acute death. We evaluated the efficacy of urtoxazumab (TMA-15, Teijin Pharma Limited), a humanized monoclonal antibody against Shiga toxin (Stx) 2 for the prevention of brain damage, dysfunction, and death in a piglet EHEC infection model. Forty-five neonatal gnotobiotic piglets were inoculated orally with 3 × 108 colony-forming units of EHEC O157:H7 strain EDL933 (Stx1⁺, Stx2⁺) when 22-24 h old. At 24 h post-inoculation, piglets were intraperitoneally administered placebo or TMA-15 (0.3, 1.0 or 3.0 mg/kg body weight). Compared to placebo (n = 10), TMA-15 (n = 35) yielded a significantly greater probability of survival, length of survival, and weight gain (p <0.05). The efficacy of TMA-15 against brain lesions and death was 62.9% (p = 0.0004) and 71.4% (p = 0.0004), respectively. These results suggest that TMA-15 may potentially prevent or reduce vascular necrosis and infarction of the brain attributable to Stx2 in human patients acutely infected with EHEC. However, we do not infer that TMA-15 treatment will completely protect human patients infected with EHEC O157:H7 strains that produce both Stx1 and Stx2.

Impact of acquisition of 16S rRNA methylase RmtB on the fitness of Escherichia coli.

The aim of this study was to elucidate the biological phenotypes of 16S rRNA methylase RmtB in Escherichia coli and the impact of RmtB acquisition on the fitness of the target bacterium. An rmtB in-frame deletion mutant in E. coli was constructed using a suicide vector (pDMS197)-based double crossover allelic exchange, and its corresponding complemented strain was established. Combined studies of microdilution susceptibility testing, conjugation experiments, growth kinetics assays, competitive experiments, biofilm formation tests and motility assays were performed to study the rmtB-mediated fitness among the prototype E. coli strain, its isogenic mutant and the corresponding complemented strain. The minimum inhibitory concentrations (MICs) of 4,6-disubstituted 2-deoxystreptamines for the rmtB wild-type strain, its isogenic mutant and the complemented strain were ≥1024, ≤2 and ≥1024mg/L, respectively. Both the growth rates and the competitive abilities of the wild-type and complemented strains were relatively inferior to the ΔrmtB mutant. There was no significant difference in biofilm formation and motility among the three strains. In conclusion, the data presented here suggest that acquisition of the 16S rRNA methylase gene rmtB in E. coli can exact a fitness cost on the bacteria, subsequently reducing the growth rate slightly and decreasing the competitive capacity of the bacterium, whereas it does not affect biofilm formation or motility.

Relationship between heat-labile enterotoxin secretion capacity and virulence in wild type porcine-origin enterotoxigenic Escherichia coli strains.

Heat-labile enterotoxin (LT) is an important virulence factor secreted by some strains of enterotoxigenic Escherichia coli (ETEC). The prototypic human-origin strain H10407 secretes LT via a type II secretion system (T2SS). We sought to determine the relationship between the capacity to secrete LT and virulence in porcine-origin wild type (WT) ETEC strains. Sixteen WT ETEC strains isolated from cases of severe diarrheal disease were analyzed by GM1ganglioside enzyme-linked immunosorbent assay to measure LT concentrations in culture supernatants. All strains had detectable LT in supernatants by 2 h of culture and 1 strain, which was particularly virulent in gnotobiotic piglets (3030-2), had the highest LT secretion level all porcine-origin WT strains tested (P<0.05). The level of LT secretion (concentration in supernatants at 6-h culture) explained 92% of the variation in time-to-a-moribund-condition (R2 = 0.92, P<0.0001) in gnotobiotic piglets inoculated with either strain 3030-2, or an ETEC strain of lesser virulence (2534-86), or a non-enterotoxigenic WT strain (G58-1). All 16 porcine ETEC strains were positive by PCR analysis for the T2SS genes, gspD and gspK, and bioinformatic analysis of 4 porcine-origin strains for which complete genomic sequences were available revealed a T2SS with a high degree of homology to that of H10407. Maximum Likelihood phylogenetic trees constructed using T2SS genes gspC, gspD, gspE and homologs showed that strains 2534-86 and 3030-2 clustered together in the same clade with other porcine-origin ETEC strains in the database, UMNK88 and UMN18. Protein modeling of the ATPase gene (gspE) further revealed a direct relationship between the predicted ATP-binding capacities and LT secretion levels as follows: H10407, -8.8 kcal/mol and 199 ng/ml; 3030-2, -8.6 kcal/mol and 133 ng/ml; and 2534-86, -8.5 kcal/mol and 80 ng/ml. This study demonstrated a direct relationship between predicted ATP-binding capacity of GspE and LT secretion, and between the latter and virulence.

Poly I:C adjuvanted inactivated swine influenza vaccine induces heterologous protective immunity in pigs.

Swine influenza is widely prevalent in swine herds in North America and Europe causing enormous economic losses and a public health threat. Pigs can be infected by both avian and mammalian influenza viruses and are sources of generation of reassortant influenza viruses capable of causing pandemics in humans. Current commercial vaccines provide satisfactory immunity against homologous viruses; however, protection against heterologous viruses is not adequate. In this study, we evaluated the protective efficacy of an intranasal Poly I:C adjuvanted UV inactivated bivalent swine influenza vaccine consisting of Swine/OH/24366/07 H1N1 and Swine/CO/99 H3N2, referred as PAV, in maternal antibody positive pigs against an antigenic variant and a heterologous swine influenza virus challenge. Groups of three-week-old commercial-grade pigs were immunized intranasally with PAV or a commercial vaccine (CV) twice at 2 weeks intervals. Three weeks after the second immunization, pigs were challenged with the antigenic variant Swine/MN/08 H1N1 (MN08) and the heterologous Swine/NC/10 H1N2 (NC10) influenza virus. Antibodies in serum and respiratory tract, lung lesions, virus shedding in nasal secretions and virus load in lungs were assessed. Intranasal administration of PAV induced challenge viruses specific-hemagglutination inhibition- and IgG antibodies in the serum and IgA and IgG antibodies in the respiratory tract. Importantly, intranasal administration of PAV provided protection against the antigenic variant MN08 and the heterologous NC10 swine influenza viruses as evidenced by significant reductions in lung virus load, gross lung lesions and significantly reduced shedding of challenge viruses in nasal secretions. These results indicate that Poly I:C or its homologues may be effective as vaccine adjuvants capable of generating cross-protective immunity against antigenic variants/heterologous swine influenza viruses in pigs.

Both enzymatic and non-enzymatic properties of heat-labile enterotoxin are responsible for LT-enhanced adherence of enterotoxigenic Escherichia coli to porcine IPEC-J2 cells.

Previous studies in piglets indicate that heat labile enterotoxin (LT) expression enhances intestinal colonization by K88 adhesin-producing enterotoxigenic Escherichia coli (ETEC) as wild-type ETEC adhered to intestinal epithelium in substantially greater numbers than did non-toxigenic constructs. Enzymatic activity of the toxin was also shown to contribute to the adhesion of ETEC and non-ETEC bacteria to epithelial cells in culture. To further characterize the contribution of LT to host cell adhesion, a nontoxigenic, K88-producing E. coli was transformed with either the gene encoding for LT holotoxin, a catalytically-attenuated form of the toxin [LT(R192G)], or LTB subunits, and resultant changes in bacterial adherence to IPEC-J2 porcine intestinal epithelial cells were measured. Strains expressing LT holotoxin or mutants were able to adhere in significantly higher numbers to IPEC-J2 cells than was an isogenic, toxin-negative construct. LT+ strains were also able to significantly block binding of a wild-type LT+ ETEC strain to IPEC-J2 cells. Adherence of isogenic strains to IPEC-J2 cells was unaltered by cycloheximide treatment, suggesting that LT enhances ETEC adherence to IPEC-J2 cells independent of host cell protein synthesis. However, pretreating IPEC-J2 cells with LT promoted adherence of negatively charged latex beads (a surrogate for bacteria which carry a negative change), which adherence was inhibited by cycloheximide, suggesting LT may induce a change in epithelial cell membrane potential. Overall, these data suggest that LT may enhance ETEC adherence by promoting an association between LTB and epithelial cells, and by altering the surface charge of the host plasma membrane to promote non-specific adherence.